miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines

miR-122, a hepato-specific microRNA (miRNA), is frequently down-regulated in human hepatocellular carcinoma (HCC). In an effort to identify novel miR-122 targets, we performed an in silico analysis and detected a putative binding site in the 3′-untranslated region (3′-UTR) of Bcl-w, an anti-apoptotic Bcl-2 family member. In the HCC-derived cell lines, Hep3B and HepG2, we confirmed that miR-122 modulates Bcl-w expression by directly targeting binding site within the 3′-UTR. The cellular mRNA and protein levels of Bcl-w were repressed by elevated levels of miR-122, which subsequently led to reduction of cell viability and activation of caspase-3. Thus, Bcl-w is a direct target of miR-122 that functions as an endogenous apoptosis regulator in these HCC-derived cell lines.     

miR-214通过调控Notch1信号对口咽鳞状细胞癌细胞株增殖、侵袭及凋亡的作用机制研究

目的研究miR-214通过调控Notch1信号对口咽鳞状细胞癌（OSCC）细胞株增殖、侵袭及凋亡的作用机制。 方法在ATCC细胞库购买OSCC细胞株,分为3组：抑制组（IN组）、模拟组（SI组）及对照组（CO组）。通过Western blot法、qRT-PCR法、TUNEL法、侵袭实验及CCK-8法分析3组癌细胞中Notch1的蛋白、mRNA表达水平、细胞凋亡、迁移及增殖能力的差异性,三组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果IN、SI、CO组细胞内miR-214mRNA表达量分别为1.15±0.26,3.34±0.89,2.08±0.67,SI组分别与IN组、CO组比较,差异具有统计学意义（t = 5.281,P = 0.002;t = 2.529,P = 0.045）;SI组、IN组的Notch1-UTR表达分别为0.42±0.11、0.86±0.09,差异具有统计学意义（t = 5.362,P = 0.006）;IN、SI、CO组的OSCC细胞凋亡率分别为（0.78±0.10）﹪、（0.32±0.06）﹪、（0.56±0.12）﹪,IN组分别与CO组、SI组比较,差异具有统计学意义（t = 3.149,P = 0.020;t = 8.823,P < 0.001）;IN、SI、CO组癌细胞的增殖率分别为（0.65±0.05）﹪、（1.26±0.06）﹪、（1.89±0.04）﹪,IN组与SI组、IN组与CO组比较,差异具有统计学意义（t = 11.056,P < 0.001;t = 27.394,P < 0.001）,CO组与SI组比较,差异具有统计学意义（t = 12.365,P < 0.001）;IN、SI、CO组癌细胞侵袭率分别为（0.13±0.02）﹪、（0.89±0.13）﹪、（0.48±0.11）﹪,SI组与CO组、SI组与IN组比较,差异具有统计学意义（t = 4.176,P = 0.006;t = 10.147,P < 0.001）;IN、SI、CO组的Notch1蛋白含量分别为：2.38±1.34、0.94±0.23、1.76±0.67,SI组与CO组、SI组与IN组比较,差异具有统计学意义（t = 2.588,P = 0.041;t = 2.368,P = 0.056）;IN、SI、CO组的Notch1 mRNA表达量分别为4.26±1.06、1.45±0.38、2.46±0.87,IN组与CO组、IN组与SI组比较,差异具有统计学意义（t = 2.935,P = 0.026;t = 5.583,P = 0.001）。 结论miR-214可能与OSCC细胞株增殖呈负相关,在细胞中miR-214含量升高,可能抑制Notch1信号通路表达,从而促进癌细胞增长、侵袭,抑制癌细胞凋亡。     

Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.     

TaqMan real-time PCR assay for specific detection of Opisthorchis viverrini DNA in Thai patients with hepatocellular carcinoma and cholangiocarcinoma

The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p = 0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.     

Identification and analysis of altered alpha1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis

Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.     

mTOR/miR-145-regulated exosomal GOLM1 promotes hepatocellular carcinoma through augmented GSK-3b/MMPs

Golgi membrane protein 1(GOLM1/GP73)is a serum marker of hepatocellular carcinoma(HCC).We have previously shown that mTOR promoted tumorigenesis of HCC through stimulating GOLM1 expression.In this study,we demonstrated that the mammalian target of rapamycin(mTOR)was a negative regulator of microRNA-145(miR-145)expression.miR-145 inhibited GOLM1 expression by targeting a coding sequence of GOLM1 gene.GOLM1 and miR-145 were inversely correlated in human HCC tissues.GOLM1-enriched exosomes activated the glycogen synthase kinase-3β/matrix metalloproteinases(GSK-3β/MMPs)signaling axis of recipient cells and accelerated cell proliferation and migration.In contrast,miR-145 suppressed tumorigenesis and metastasis.We suggest that mTOR/miR-145/GOLM1 signaling pathway should be targeted for HCC treatment.     

Rock2 promotes the invasion and metastasis of hepatocellular carcinoma by modifying MMP2 ubiquitination and degradation

Rho-associated coiled-coil-containing protein kinase 2 (Rock2) is a downstream effector of Rho that plays an important role in the tumorigenesis and progression of hepatocellular carcinoma (HCC). Matrix metalloproteinase 2 (MMP2) is a master regulator of tumor metastasis. In this study, we investigated the collections of Rock2 and MMP2 in HCCs and determined the potential role and molecular mechanism of Rock2 in MMP2-mediated invasiveness and metastasis. We found that Rock2 and MMP2 were markedly overexpressed in HCCs compared with the corresponding adjacent tissues, where a positive correlation in their expression was found. The knockdown of Rock2 significantly decreased MMP2 expression and inhibited the invasion and metastasis of HCC in vitro and in vivo. Additionally, the upregulation of MMP2 rescued the decreased migration and invasion induced by the knockdown of Rock2, whereas the knockdown of MMP2 decreased Rock2-enhanced HCC migration and invasion. Mechanistically, Rock2 stabilized MMP2 by preventing its ubiquitination and degradation. Together, our results link two drivers of invasion and metastasis in HCC and identify a novel pathway for MMP2 control.     

Epigenetic inactivation of SLIT2 in human hepatocellular carcinomas

Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene. In addition, hypermethylation of its promoter region has been detected in various cancers, including breast and lung cancer, colorectal carcinoma, and gliomas. Here, we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma (HCC). Downregulation of SLIT2 was detected in 6 of 8 (75%) HCC cell lines by quantitative real-time RT-PCR (qRT-PCR), and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore, expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC). Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples (83.3%), and the decrease in expression was significantly correlated with CpG island hypermethylation. This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC. Moreover, overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth, migration, and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC. Thus, SLIT2 may be useful as a therapeutic target in the treatment of HCC.     

Regulation of human hepatocellular carcinoma cells by Spred2 and correlative studies on its mechanism

Members of the Spred gene family are negative regulators of the Ras/Raf-1/ERK pathway, which has been associated with several features of the tumor malignancy. However, the effect of Spred genes on hepatocellular carcinoma (HCC) remains uninvestigated. In the present work, we analyzed the in vitro and in vivo effects of Spred2 expression on the hepatic carcinoma cell line, SMMC-7721. In addition to attenuated ERK activation, which inhibited the proliferation and migration of unstimulated and HGF-stimulated SMMC-7721 cells. Adenovirus-mediated Spred2 overexpression induced the activation of caspase-3 and apoptosis, as well as reduced the expression level of Mcl-1. Most importantly, the knockdown of Spred2 markedly enhanced tumor growth in vivo. In conclusion, these results suggest that Spred2 could qualify as a potential therapeutic target in HCC.     

Network analysis and proteomic identification of vimentin as a key regulator associated with invasion and metastasis in human hepatocellular carcinoma cells

Poor prognoses have long been associated with the high relapse and metastasis of human hepatocellular carcinoma (HCC). To achieve long-term survival, it is necessary to identify new HCC biomarkers and investigate their roles in cell mobility and invasiveness. Of note, overexpression of vimentin (Vim) was significantly correlated with tumor nuclear grade (p=0.01) and the invasive potential, indicating that Vim may be a promising candidate in regulating HCC metastasis. RNA interference-mediated silencing of Vim (siVim) suppressed the invasive and migratory propensity, and matrix metalloproteinase (MMP)-9 activity, and elicited morphological changes in poorly differentiated SK-Hep-1 cells. Moreover, we performed a comprehensive proteomic analysis to survey global protein changes mediated by siVim in SK-Hep-1 cells. Significant changes in cytoskeleton protein but not messenger RNA levels encoding these targeted proteins were observed. All of the data in the current study and a network analysis implied that abolition of Vim may disturb the expression and stability of various cytoskeletal proteins through promoting the ubiquitin system, resulting in impaired cell adhesion and motility. Collectively, an integrated approach represents a modality to explore novel relationships in a proteome complex and highlights the functional roles of Vim in HCC metastasis. This article is part of a Special Issue entitled: Translational Proteomics.     

lncRNA OSER1-AS1 acts as a ceRNA to promote tumorigenesis in hepatocellular carcinoma by regulating miR-372-3p/Rab23 axis

Long non-coding RNAs (lncRNAs) are crucial regulators of tumorigenesis and progression in human cancer, including hepatocellular carcinoma (HCC). However, the role of most lncRNAs that are dysregulated in HCC remains to be elucidated. Here, we investigated the role of OSER1-AS1 in the progression of HCC. The results of database and qRT-PCR analysis demonstrated that OSER1-AS1 was highly expressed in HCC tissues and the high expression of OSER1-AS1 was closely associated with larger tumor size, advanced tumor stages, lower disease free survival and overall survival of HCC patients. OSER1-AS1 knockdown significantly inhibited the proliferation, invasion and migration of HCC cells, and induced the apoptosis. In addition, the dual luciferase reporter assay directly demonstrated that OSER1-AS1 functioned as a molecular sponge for miR-372-3p to promote Rab23 expression. Moreover, the results of immunohistochemistry and western blot analysis showed that Rab23 was highly expressed in HCC tissues, and the high expression of Rab23 was closely associated with the poor overall survival of HCC patients. Immunofluorescence assay also found the subcellular localization of Rab23 in HCC cells. Rab23 was obviously downregulated in cells that were transfected with miR-372-3p mimics. MiR-372-3p mimics significantly inhibited the proliferation and invasion of HCC cells). Rab23 restoration partially reversed miR-372-3p-induced tumor suppressive effects on HCC cells. In conclusion, we found that OSER1-AS1 acted as a ceRNA to sponge miR-372-3p, thereby positively regulating the Rab23 expression and ultimately acting as a tumor suppressor gene in HCC progression.     

RASSF1A Ala133Ser polymorphism is associated with increased susceptibility to hepatocellular carcinoma in a Turkish population

Aim

The tumor suppressor gene Ras association domain family 1 isoform A (RASSF1A) regulates cell cycle regulation, apoptosis and microtubule stability and is inactivated by promoter hypermethylation at a high frequency in hepatocellular carcinoma (HCC). A guanine (G)/thymine (T) common single nucleotide polymorphism (SNP) at first position of codon 133 in RASSF1A gene determines an alanine (Ala) to serine (Ser) (Ala133Ser) amino acidic substitution which may alter cancer risk by influencing the function of RASSF1A protein.

Methods

To determine the association of the RASSF1A Ala133Ser polymorphism with the risk of HCC development in a Turkish population, a hospital-based case–control study was designed consisting of 236 subjects with HCC and 236 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the RASSF1A Ala133Ser polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.

Results

Allele and genotype associations of RASSF1A Ala133Ser polymorphism with HCC susceptibility were observed in comparisons between the patient and control samples (P < 0.001). Risk of HCC development in this Turkish population was significantly increased in carriers of the Ser133 variant allele of Ala133Ser polymorphism (Ala/Ser and Ser/Ser genotypes) when compared with homozygote Ala/Ala genotype (OR = 5.47, 95% CI = 3.63–8.25, P = 0.001).

Conclusion

Because our results suggest for the first time that the Ser133 allele of RASSF1A Ala133Ser polymorphism may be a genetic susceptibility factor for HCC in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins.     

IGF2 polymorphisms are associated with hepatitis B virus clearance and hepatocellular carcinoma

The aim of this study was to determine whether IGF2 polymorphisms are associated with the clearance of hepatitis B virus (HBV) infection and the risk of hepatocellular carcinoma (HCC). A total of 1095 Korean subjects were prospectively enrolled in this case-control study. The rates of IGF2 polymorphisms were determined in each group. The IGF2+820G allele (IGF2+820G/G) and the IGF2+6815A/A genotype were strongly associated with the resolution of HBV infection (OR=0.62-0.73; P=0.001-0.03 and OR=0.71; P=0.03, respectively). Haplotype analysis showed that IGF2-haplotype5 (A-C-C-T-A-T-G) and IGF2-haplotype1 (T-C-T-T-A-C-A) were significantly associated with the clearance and persistence of HBV infection (OR=0.55-0.58, P=0.009-0.01 and OR=1.31-1.65, P=0.001-0.007, respectively). On the other hand, the IGF2+2482C/C or +820G/G genotypes were significantly associated with a higher risk of HCC (OR=1.88, 1.68; P=0.04). IGF2 polymorphisms were found to be strongly associated with the clearance of HBV or the occurrence of HCC in patients with chronic HBV infection.     

Methylation-mediated miR-214 regulates proliferation and drug sensitivity of renal cell carcinoma cells through targeting LIVIN

LIVIN, a member of the inhibitor of apoptosis proteins (IAPs), is reported playing important roles in the development and progression of multiple human cancers. However, its underlined mechanisms in human renal cell carcinoma (RCC) are still needed to be clarified. In the present study, we reported that inhibition of miR-214 promoted the expression of LIVIN, then facilitated RCC cells growth and reduced the sensitivity of RCC cells to chemotherapeutic drugs. In constant, overexpression of miR-214 had contradictory effects. Further investigation showed that miR-214 was down-regulated in RCC because of abnormal methylation. In addition, DNA methyltransferase DNMT1, miR-214 and LIVIN are directly correlated in RCC patients. In conclusion, these results suggest that abnormal miR-214 methylation negatively regulates LIVIN, which may promote RCC cells growth and reduced the sensitivity of RCC cells to chemotherapeutic drugs.     

Tumor-suppressive microRNA-10a inhibits cell proliferation and metastasis by targeting Tiam1 in esophageal squamous cell carcinoma

Aberrant microRNAs (miRNAs) expressions could contribute to the progression of numerous cancers, including esophageal squamous cell carcinoma, while miR-10a participates in multiple biological processes on cancers. However, the molecular mechanism of miR-10a in esophageal squamous cell carcinoma (ESCC) has not been investigated. Herein, miR-10a was significantly reduced in ESCC clinical tissues and ESCC cell lines (EC109 and TE-3). In addition, immunohistochemistry indicated that the expressions of α-SMA, Ki-67, and PCNA in tumor tissues were higher than that of controls. In vitro, overexpression of miR-10a dramatically suppressed cell proliferation and enhanced cell apoptosis, while the decrease of miR-10a expressed the opposite outcome. Specially, overexpression of miR-10a caused a G0/G1 peak accumulation. Moreover, miR-10a also negatively regulated ESCC cell migration and invasion. Furthermore, targetscan bioinformatics predictions and the dual-luciferase assay confirmed that Tiam1 was a direct target gene of miR-10a. The statistical analysis showed Tiam1 was negatively in correlation with miR-10a in ESCC patient samples. And silencing Tiam1 could lead to a decline on cell growth, invasion, and migration in ESCC cell lines, while it could enhance cell apoptosis and cause a G0/G1 peak accumulation. In vivo, it revealed that miR-10a notably decreased the tumor growth and metastasis in xenograft model and pulmonary metastasis model. And it showed a lower expressions of Tiam1 in the miR-10a mimics group by immunohistochemistry. Taken together the results, they indicated that miR-10a might function as a novel tumor suppressor in vitro and in vivo via targeting Tiam1, suggesting miR-10a to be a candidate biomarker for the ESCC therapy.